The present invention relates to methods, reagents, and reagent kits for detecting discrete functional conformations of proteins concurrently in individual cells, particularly by flow cytometry. In particular, the invention relates to methods, reagents, and reagent kits for the determination of the phosphorylation status of the retinoblastoma susceptibility gene protein (pRB) in individual cells using multiparameter flow cytometry.
The recent revolution in genomics technology, and in particular, the development of high density nucleic acid microarrays, has focused extraordinary attention on the differential expression of genes as markers of cellular differentiation, prognosticators of disease, and potential targets for interventional therapy. Brown et al., Nature Genet. 21 (Suppl.):33-37 (1999); Duggan et al., Nature Genet. 21 (Suppl.):10-14 (1999); Cole et al., Nature Genet. 21 (Suppl.):38-41 (1999); Debouck et al., Nature Genet. 21 (Suppl.):48-50 (1999). Although an understanding of the cell""s transcriptional program is indeed important to all of these goals, the function of many critical proteins is regulated, at least in part, at the posttranslational level, a level to which transcription-based approaches are perforce indifferent.
One such critical protein is that encoded by the retinoblastoma susceptibility gene (pRB; pRb), which plays a pivotal role in the regulation of the cell cycle. pRB restrains cell cycle progression by maintaining a checkpoint in late G1 that controls commitment of cells to enter S phase. The critical role that pRB plays in cell cycle regulation explains its status as archetypal tumor suppressor: loss of pRB function results in an inability to maintain control of the G1 checkpoint; unchecked progression through the cell cycle is, in turn, a hallmark of neoplasia.
pRB activity is controlled by changes in phosphorylation. pRB is hypophosphorylated in normal quiescent cells (in G0 phase) and in cells that are in early G1. With continued progression through G1, cyclin-dependent kinases. (Cdk; Cdk), in association with their respective cyclins, phosphorylate pRB at a number of serine and threonine residues. Unphosphorylated, pRB binds to and sequesters transcription factors of the E2F family. Phosphorylated, pRB discharges these factors, the factors in turn activating transcription of genes coding for proteins regulating DNA replication and cell proliferation. These events commit the cell to entry into S phase. Later, in late mitosis, type 1 protein phosphatases dephosphorylate pRB, restoring the active, E2F-sequestering form, thus resetting the cycle.
pRB is also essential in the terminal differentiation of cells of various lineages. During terminal differentiation, when cells exit the cycle, pRB expression is upregulated and the protein remains in the activexe2x80x94that is, hypophosphorylatedxe2x80x94state. Mice homozygously deleted for the RB gene show defective differentiation of various tissues.
Given the critical role that pRB phosphorylation plays in controlling progression of cells through the cell cycle and in mediating terminal differentiation, there exists a need for assays that permit the ready determination of pRB phosphorylation status.
Typically, the phosphorylation status of pRB is assayed in vitro, measuring 32P-labeling and/or electrophoretic mobility of the protein after isolation and identification by Western blotting. The procedure is cumbersome, and more importantly risks artifactual activation of phosphatases that may dephosphorylate the protein during or after cell lysis. There thus exists a need for methods of measuring pRB phosphorylation in intact cells.
Furthermore, the existing methods measure phosphorylation of pRB in bulk culture. Several questions regarding the mechanism by which pRB controls cell cycle progression cannot be answered using such assays. For example, is phosphorylation of pRB within the cell an all-or-none phenomenon, or is there instead a mixture of hypophosphorylated and hyperphosphorylated pRB molecules at varying proportions throughout the cycle? What proportion of pRB molecules is phosphorylated within the cell during G1, prior to entrance to S phase? Is there a critical threshold in the ratio of hypophosphorylated to hyperphosphorylated pRB molecules that determines the transition of cells to quiescence or to commitment to enter S? Is it the ratio of hypo- to hyperphosphorylated pRB or, rather, the total level of the latter that is critical for cell commitment to enter S phase?
Study of the average behavior of cells in bulk culture also precludes evaluation of heterogeneity in the cycling of individual cells in the population. Such heterogeneity in cell cycle kinetics in tumor cell populations is recognized as a major impediment to successful therapy of cancer. There thus exists a need for methods that permit the heterogeneity of cell cycle kinetics to be assayed, and a particular need for methods that would permit cell cycle heterogeneity to be assessed in populations of tumor cells.
The heterogeneity in the cycling kinetics and timing of cells in culture typically obligates artificial synchronization of the cells in culture to permit meaningful results to be obtained using the existing bulk assays; and yet this cell cycle synchronization, when induced by inhibitors of DNA polymerase, is associated with growth imbalance and unscheduled expression of cyclins. Gong et al., Cell Growth Differ. 6:1485-1493 (1995). There thus exists a need for methods that permit the phosphorylation status of pRB to be measured in individual cells without exogenous intervention in the cell cycle.
Methods permitting the phosphorylation status of pRB to be measured in individual cells would prove useful additionally in identifying and characterizing antiproliferative agents that act by halting progression through the cell cycle.
ONCONASE(copyright), initially named protein P30, is a basic protein of 12,000 MW isolated from oocytes or early embryos of Rana pipiens. ONCONASE(copyright) shows antiproliferative activity in vitro, suppressing proliferation of tumor cell lines of various lineages, including those of hematological origin. ONCONASE(copyright) has also been shown to inhibit growth of certain tumors in vivo in mice.
Although ONCONASE(copyright) is currently in clinical trials for treatment of patients with advanced pancreatic adenocarcinoma and malignant mesothelioma, the mechanism of its antitumor activity is still poorly understood. The protein is known to have both cytostatic and cytotoxic effects, the former manifesting as an increase in the proportion of cells in G1 phase of the cell cycle; but the mechanism by which the drug effects such cell cycle arrest is unknown. It would be advantageous to have an assay that would permit such a drug""s effects on the cell cycle, and in particular, its effect, if any, on pRB phosphorylation, readily to be assayed on a single-cell basis.
Reliable measures of pRB phosphorylation status in individual cells would permit pRB to serve as a marker for distinguishing quiescent from cycling cells. Although certain cell features, such as cellular RNA content, nucleolar mass, chromatin structure (degree of chromatin condensation), expression of the Ki-67 antigen, or expression other proliferation-associated proteins have been proposed as markers distinguishing cycling from noncycling cells, there is as yet no generally accepted, easily measurable marker which discriminates G0 from G1 cells. There thus exists a need in the art for a marker that reliably discriminates quiescent cells from cycling cells.
Recently, two mAbs recognizing human pRB have been described, one of which specifically detects the underphosphorylated form of this protein (pRBPxe2x88x92), the other of which reacts with total pRB, regardless of phosphorylation state (pRBT). Dunaief et al., Cell 79:119-130 (1994); Wang et al., Oncogene 8:279-288 (1993); Terada et al., J. Immunol. 147:698-704 (1991); Zarkowska et al., Oncogene 14:249-266 (1997). These antibodies have been used to study several aspects of pRB metabolism in bulk culture. There exists a need to adapt these antibodies to methods permitting detection of pRB phosphorylation states in intact cells on a single-cell basis.
The present invention solves these and other problems in the art by presenting methods and reagents that permit the concurrent and discriminable detection of discrete functional conformations of proteins within a single cell. In particular, the invention provides methods and reagents for the flow cytometric determination of multiple pRB phosphorylation states in individual cells.
We have demonstrated, for the first time, that anti-pRB antibodies that distinguish the phosphorylation state of pRB may successfully be conjugated to fluorophores (fluorochromes) without loss of specificity; that when conjugated to such fluorophores, these antibodies provide sufficient signal to permit detection of pRB in individual cells; that when simultaneously applied to cells that have been fixed and permeabilized, these antibodies bind to their respective functional conformations of pRB within the cell without mutual interference; and that when conjugated to flow cytometrically distinguishable fluorophores, these antibodies permit the concurrent detection of discrete functional conformations (phosphorylation states) of pRB to be detected in single cells.
Using these fluorophore-conjugated antibodies with dyes that bind stoichiometrically to DNA, we have found that the phosphorylation status of pRB may now be correlated with the cell""s position in the cell cycle, without the need for artificial synchronization of the cycle. When further used with antibodies specific for other protein components of the cell cycle machineryxe2x80x94such as cyclins, cyclin dependent kinases, and Cdk inhibitorsxe2x80x94the methods permit complex intracellular interactions of pRB to be assessed.
Thus, in a first aspect, the present invention provides a method for determining the relative intracellular conformational states of a protein, comprising: contacting a cell with a first antibody, said first antibody specific for a first conformation of said protein, and a second antibody, said second antibody specific for at least one other conformation of said protein, said first and second antibodies being distinguishably labeled; detecting the binding of each of said antibodies concurrently by said cell; and determining the relative binding thereof.
Where the conformational state of the cell may change rapidly, the method may further comprise the antecedent step of fixing the cell. Where the protein is internal to the cellxe2x80x94either cytoplasmic or nuclearxe2x80x94the method further comprises the step, before flow cytometric detection, of permeabilizing the cell.
In preferred embodiments, the antibodies are labeled with fluorophores and the fluorophores are distinguishable by a laser cytometer. Where the laser cytometer is a flow cytometer, the fluorophores are flow cytometrically distinguishable. The fluorophores may conjugated directly or indirectly to the antibodies, with the direct conjugation of at least one, preferably at least two, antibodies presently preferred. Although any fluorophore that permits laser cytometric detection may usefully be employed, those presently preferred are selected from the group consisting of: FITC, PE, PerCP, APC, PE-CY5 tandem fluorophore and PerCP-CY5.5 tandem fluorophore.
In another aspect, the present invention provides a method for determining the relative intracellular conformational states of pRB, comprising: contacting a cell with a first antibody, said first antibody specific for a first conformation of pRB, and a second antibody, said second antibody specific for at least one other conformation of pRB, said first and second antibodies being distinguishably labeled;
1. A method for determining the relative intracellular conformational states of a protein, comprising:
contacting a cell with a first antibody, said first antibody specific for a first conformation of said protein, and a second antibody, said second antibody specific for at least one other conformation of said protein, said first and second antibodies being distinguishably labeled; detecting the binding of each of said antibodies concurrently by said cell; and then determining the relative binding thereof.
In preferred embodiments of this aspect of the invention, each of the pRB conformations is correlated with a discrete phosphorylation state of the protein. In one such embodiment, the first antibody is specific for a conformation assumed by the hypophosphorylated form of pRB, and the second antibody is specific for at least one other conformation of pRB; this may include specificity for all functional conformations of pRB. In another embodiment, the first antibody is specific for all conformations of pRB, and the second antibody is specific for a subset thereof.
The methods of this second aspect of the invention may further comprise the step of contacting said cell with a fluorescent nucleic acid stain, and then detecting, preferably by laser cytometry, most preferably by flow cytometry, the binding to DNA of said stain concurrently with detecting the binding to pRB of said first and second antibody.
The methods of this aspect may further comprise contacting said cell with a third antibody, said third antibody being specific for a second protein and distinguishable from each of said first and second antibodies, and then detecting the concurrent binding of each of said antibodies to said cell. In preferred embodiments, the second protein may be a cyclin, a cyclin dependent kinase, or a cyclin dependent kinase inhibitor.
In another aspect, the invention provides methods of assaying, or screening, compounds for antiproliferative activity, comprising: contacting a sample of proliferating cells with said compound in vitro; contacting said cells with a first antibody specific for a conformation assumed by the hypophosphorylated form of pRB and a second antibody, said second antibody specific for at least one other conformation of pRB and being flow cytometrically distinguishable from said first antibody; flow cytometrically detecting the binding of each of said antibodies concurrently by said cell; wherein an increased ratio of hypophosphorylated pRB to total pRB indicates antiproliferative activity of said compound.
In a further aspect, the invention provides methods of assessing the in vivo antiproliferative effect of a compound, comprising: contacting a sample of cells obtained from a subjectxe2x80x94after the in vivo administration of said compound to said subjectxe2x80x94with a first antibody, said first antibody specific for a conformation assumed by the hypophosphorylated form of pRB and a second antibody, said second antibody specific for at least one other conformation of pRB and flow cytometrically distinguishable from said first antibody; and flow cytometrically detecting the binding of each of said antibodies concurrently by said cell; wherein an increased ratio of hypophosphorylated pRB to total pRB, as compared to the ratio obtained from cells identically assayed that were obtained prior to administration of said agent, indicative of in vivo antiproliferative effect.
In yet a further aspect, the invention also provides methods of assessing the proliferative potential of a heterogeneous population of cells, comprising: contacting said cells with a first antibody specific for a conformation assumed by the hypophosphorylated form of pRB and a second antibody, said second antibody specific for at least one other conformation of pRB and flow cytometrically distinguishable from said first antibody; and then flow cytometrically detecting the binding of each of said antibodies concurrently by said cell; wherein cells with a decreased ratio of hypophosphorylated pRB to total pRB are determined to have increased proliferative potential.
In another aspect, the invention provides reagent kits that facilitate the practice of the subject methods. Thus, in one embodiment, the invention provides a kit for detecting the phosphorylation status of pRB in individual cells, comprising: a first antibody, said first antibody specific for a first phosphorylation state of pRB; and a second antibody, said second antibody specific for at least one other phosphorylation state of pRB; wherein said first and second antibodies are flow cytometrically distinguishable from one another. In preferred embodiments, the first antibody is specific for the hypophosphorylated form of pRB and the second antibody is specific for total pRB. In embodiments particularly suited to flow cytometric analysis, each of said antibodies is conjugated to a fluorophore, and said fluorophores are flow cytometrically distinguishable from one another.